Download AlphaScreen SureFire User Guide

AlphaScreen® SureFire®
Cellular Kinase Assays
User Guide
For Laboratory Use Only
RESEARCH REAGENTS FOR RESEARCH PURPOSES ONLY
INTRODUCTION ....................................................................................................................... 3
ALPHASCREEN® SUREFIRE® ASSAY PRINCIPLE............................................................... 4
KIT CONTENTS ........................................................................................................................ 5
CONTROL LYSATES.................................................................................................................. 5
REACTION BUFFER ................................................................................................................... 5
LYSIS BUFFER ......................................................................................................................... 6
ACTIVATION BUFFER ................................................................................................................ 6
DILUTION BUFFER .................................................................................................................... 6
PRECIPITATION ........................................................................................................................ 6
SHELF LIFE ............................................................................................................................. 6
TARGET SELECTION ............................................................................................................ 10
COMMON PROTOCOLS ........................................................................................................ 12
GENERAL TRANSFER PROCEDURE .......................................................................................... 12
SINGLE WELL PROCEDURES ................................................................................................... 13
SINGLE REAGENT ADDITION VS DUAL REAGENT ADDITION PROCEDURES .................................... 14
ASSAY OPTIMIZATION ......................................................................................................... 15
SELECTION OF ASSAY FORMAT ............................................................................................... 15
CELL SEEDING AND DENSITY ................................................................................................... 15
TIMECOURSE OF ACTIVATION .................................................................................................. 15
ASSAY BUFFER SYSTEM & DMSO TOLERANCE ........................................................................ 16
MAXIMAL RESPONSE WINDOW AND BASAL PHOSPHORYLATION .................................................. 16
TROUBLESHOOTING ............................................................................................................ 18
LOW COUNTS ........................................................................................................................ 18
HIGH BACKGROUND ............................................................................................................... 18
POOR ASSAY SENSITIVITY...................................................................................................... 19
POOR CELL STIMULATION ...................................................................................................... 19
DAY TO DAY VARIATION ......................................................................................................... 19
FAQS....................................................................................................................................... 20
CONTACT DETAILS............................................................................................................... 22
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Introduction
The AlphaScreen® SureFire® range of cellular kinase assays have been
designed to directly measure endogenous levels of phosphorylated proteins in
a cell-based format. Combined with PerkinElmer’s versatile and highly
sensitive AlphaScreen® technology, the SureFire® cellular kinase assays
provide an ideal, no-wash solution for measuring intracellular signaling
events.
Flexible Homogeneous Assays
AlphaScreen SureFire cellular kinase assays measure endogenous levels of
cellular proteins, and so can be used with many different types of cells,
including primary cells, provided the cells have an active signaling pathway
and express the particular protein of interest at sufficient levels. These assays
can also be used on cell lines expressing transfected kinases or whole-protein
biochemical assays.
There are several applications for detection of intracellular kinase activation.
Assays can be used to monitor modulators of cellular receptors, including G
protein-coupled receptors (GPCRs), cytokine receptors and receptor tyrosine
kinases (RTKs), through specific agonist-mediated activation of intracellular
kinase pathways. The SureFire range of cellular kinase assays includes
targets activated by RTK signaling via PI3-kinase pathway signaling or MAPKmediated pathways (i.e. insulin signaling), inflammatory receptor signaling via
MAPK, TGFβ or NFκB phosphorylation, and cytokine receptor activation
through JAK/STAT phosphorylation. The list of assays is rapidly expanding,
and the latest product offerings can be found at the Perkin Elmer website
(las.perkinelmer.com).
HTS compatible
Whether you are running single plates or hundreds of plates, AlphaScreen
SureFire assays will work for you. These two advanced technologies provide
the ultimate homogeneous non-radiometric assay for full-length activated
phosphorylated kinases in cell lysates. Easily miniaturizable, AlphaScreen
SureFire is the choice for rapid and accurate high throughput screening.
For rapid detection of full-length activated kinases in cell lysates — even with
difficult targets…
AlphaScreen SureFire Cellular Kinase Assays
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AlphaScreen® SureFire® Assay
Principle
The SureFire family of optimized cellular kinase assays detect phosphorylated
proteins by immunosandwich capture. One antibody is directed against a
specific phospho-epitope on the target protein, while the other is directed
against an epitope on a distal part of the protein. The distal antibody binds the
target protein in cell lysates, independent of the phosphorylation state of the
protein. However, the phospho-antibody can only bind when the phosphoepitope on the protein is formed – usually by a specific signaling cascade.
Therefore, the amount of immuno-sandwich complexes that are formed is
dependent on the amount of phosphorylated target in the cellular lysates.
SureFire assays are specifically designed to work with the AlphaScreen
General IgG (Protein A) beads. AlphaScreen is a proximity-based technology
that only emits a signal when donor and acceptor beads are brought into
close proximity. Each antibody in the SureFire kits is specifically selected
such that it will only bind either the donor or acceptor bead. Thus, only
immuno-complexes that contain both antibodies can bind both beads, and are
subsequently detected. The assay is efficient at measuring both agonist and
antagonist activities that affect protein phosphorylation.
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Kit Contents
The SureFire kits are comprised of several proprietary components designed
for optimal performance across a number of cellular systems. The kits contain
the following components:
•
•
•
•
•
Control lysates
Reaction buffer
Lysis buffer
Activation buffer
Dilution buffer (only supplied with 2-step kits)
The amounts and recommended handling of each of the components that are
supplied with each kit is detailed in Tables 1, 2, 3 and 4.
SureFire kits are designed to work with the AlphaScreen® General IgG
(Protein A) Detection Kit (Catalogue 6760617M, 6760617R and 6760617C).
These AlphaScreen products are supplied separately, and so need to be
ordered along with the SureFire kits. When ordered together, these
companion products are usually delivered together, but are in separate boxes.
All kits are available in 500 pt, 10,000 pt and 50,000 pt pack sizes.
Control lysates
A small quantity of lyophilized cell lysates are included with each kit, and are
intended for use as a control to ensure that all kit reagents are working as
expected. Negative controls are cell lysates prepared from flasks of
unstimulated, or sometimes inhibited, cells that contain low amounts of the
phophorylated analyte. Positive controls are cell lysates prepared from flasks
of cells stimulated with an agonist to promote phosphorylation of the
phosphoprotein of interest. The cells used to prepare the control lysates vary
from kit to kit, and are chosen because they express detectable levels of the
phosphoprotein of interest. The signal that is achieved with these may be
quite different from that observed in other cell lines, and is mainly dependent
on expression levels in the cell line of interest.
Reaction buffer
Supplied at working concentration, the SureFire Reaction buffer is a
proprietary mixture of antibodies and buffer components, pre-diluted to an
optimal concentration for detecting cellular phosphoproteins in combination
with the AlphaScreen Protein A IgG detection kits, which are sold separately
by Perkin Elmer. As this reagent contains all of the antibodies required for the
assay, this solution must be stored correctly at 4ºC, and not frozen. Vortexing
is unnecessary and should also be avoided.
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Lysis Buffer
Supplied as a 5X concentrate, the SureFire Lysis buffer is a proprietary
mixture of buffers, detergents, and phosphatase inhibitors, optimised for lysis
of a broad range of cells without releasing nuclear DNA. Additives can be
supplemented to the Lysis buffer as required for particular cells, and may
include excipients such as protease inhibitors or extra detergents. These will
need to be checked on a case by case basis to ensure that they do not
interfere with the assay.
Activation buffer
Supplied at working concentration, the SureFire Activation buffer is a
proprietary formulation that acts to enhance the sensitivity of detection of
many cellular phosphoproteins. Components of this reagent WILL precipitate
during normal storage at 4ºC, and so this reagent MUST BE REDISSOLVED
PRIOR TO EVERY USE. Often the easiest way to achieve this is to incubate
Activation buffer in a 37ºC water bath for 5-10 minutes, and then mix
thoroughly by vortex or immersion. If this is inconvenient, this reagent is quite
stable and can be readily stored at room temperature. If precipitates still form,
simply re-dissolve as described above.
Dilution buffer
Supplied at working concentration, the SureFire Dilution buffer is used to
dilute AlphaScreen donor beads, and is only required for 2-step SureFire
assay procedures. It is not supplied with 1-step assay protocols as it is not
required. It is strongly recommended that this component is always stored
correctly at 4ºC.
Precipitation
Any precipitates that form in 5X Lysis buffer or Activation buffer can be redissolved by warming to 37ºC and mixing. If precipitates form in the Reaction
buffer, it should be discarded.
Shelf Life
All components should be stored as indicated until the expiry date stated. Kits
have a guaranteed shelf life of between 6-9 months from the date of
manufacture.
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Table 1: Listing of components in SureFire® assay 1-step kits
Kit size
500 Point
10000 Point
50000 Point
Lysis Buffer (5X)
5 X 2 mL
4 X 60 mL
3 X 400 mL
Activation Buffer
1 X 2 mL
1 X 60 mL
1 X 300 mL
Reaction Buffer
2 X 1.7 mL
2 X 35 mL
1 X 360 mL
Negative Cell Lysate
50 µL
250 µL
250 µL
Positive Cell Lysate
50 µL
250 µL
250 µL
Assay Control Samples (lyophilized)
Table 2: Listing of components in SureFire® assay 2-step kits
Kit size
500 Point
10000 Point
50000 Point
Lysis Buffer (5X)
5 X 2 mL
4 X 60 mL
3 X 400 mL
Activation Buffer
1 X 2 mL
1 X 60 mL
1 X 300 mL
Reaction Buffer
2 X 1.3 mL
1 X 45 mL
1 X 225 mL
Dilution Buffer
1 X 1.5 mL
1 X 25 mL
2 X 60 mL
Negative Cell Lysate
50 µL
250 µL
250 µL
Positive Cell Lysate
50 µL
250 µL
250 µL
Assay Control Samples (lyophilized)
*Also required for running SureFire assays: Protein A general IgG detection
kit. Available from Perkin Elmer in 500pt (Cat.# 6760617C), 10,000pt (Cat.#
6760617M), and 50,000pt (Cat.# 6760617R) pack sizes.
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Table 3: Buffer preparation and Storage Conditions for 1-step assay kits
Storage and preparation descriptions
5X Lysis buffer
Store 5X Lysis buffer at 4oC. After dilution, excess 1X
Lysis buffer can be frozen and thawed up to 5 times
without loss in activity.
Activation buffer
Precipitation may occur at 4oC. Warm slowly to 37oC
and gently mix to re-suspend. Activation buffer can be
stored at room temperature with no loss in activity.
Reaction buffer
Keep at 4oC while in use. Do not freeze.
Once diluted discard unused reaction buffer.
AlphaScreen® Protein A IgG Kit
Store at 4oC in the dark.
Reaction buffer + Activation buffer and
AlphaScreen® beads
Mix Reaction buffer (60 parts), Activation Buffer (10
parts) and Donor and Acceptor beads (1 part each). Mix
to be stored at room temperature and used the same
day, excess mix should be discarded.
Assay control samples
Negative cell lysate
Stable while lyophilized at 4oC for life of kit.
Reconstitute lysates in water for 15 min at room
temperature and mix by pipetting. Once reconstituted,
lysates should be frozen at -20oC in single use aliquots.
Positive cell lysate
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Table 4: Buffer preparation and Storage Conditions for 2-step assay kits
Storage and preparation descriptions
5X Lysis buffer
Store 5X Lysis buffer at 4ºC. After dilution, excess 1X
Lysis buffer can be frozen and thawed up to 5 times
without loss in activity.
Activation buffer
Precipitation may occur at 4ºC. Warm slowly to 37ºC
and gently mix to re-suspend. Activation buffer can be
stored at room temperature with no loss in activity.
Reaction buffer
Keep at 4ºC while in use. Do not freeze.
Once diluted discard unused reaction buffer.
AlphaScreen® Protein A IgG Kit
Store at 4ºC in the dark.
Reaction buffer + Activation buffer and
AlphaScreen® Acceptor beads
Mix Reaction buffer (40 parts), Activation Buffer (10
parts) and Acceptor beads (1 part). Mix to be stored at
room temperature and used the same day, excess mix
should be discarded.
Dilution buffer + AlphaScreen® Donor
beads
Mix Dilution buffer (20 parts) and Donor beads (1 part).
Mix to be stored at room temperature and used the
same day, excess mix should be discarded.
Assay control samples
Negative cell lysate
Stable while lyophilized at 4oC for life of kit.
Reconstitute lysates in water for 15 min at room
temperature and mix by pipetting. Once reconstituted,
lysates should be frozen at -20oC in single use aliquots.
Positive cell lysate
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Target Selection
The SureFire range of assays can be used to assay for a number of cellular
pathways, including:
•
•
•
•
•
•
•
•
MAPK signaling
Akt signaling
Translational control
NFκB signaling
TGFβ/Smad signaling
Apoptosis
Tyrosine kinase receptor signaling
GPCR signaling
For each pathway, there are multiple SureFire kits that are available, each
measuring a different target or phosphorylation site. This allows signaling
pathways to be analysed in many cell lines, even if particular kinases in a
signaling pathway are not well expressed. Furthermore, because of the low
amounts of cell lysate used in the SureFire assays, important information
regarding specificity and off-target compounds effects can be deduced from a
single cell culture well through measurement of several target proteins from
the same lysate.
Cytokine
receptors
GPCRs
Growth factor
receptors
Inflammatory
receptors
β-arrestin
Jak/Stat signaling
i.e. Stat-3, Stat-5
Stress/inflammation
signaling pathways i.e.
NFκB, p38 MAPK, Jnk
Ca2+
cAMP
IP1
PI3-kinase/mTOR signaling
i.e. PDK-1, Akt, p70S6k, 4EBP1, S6 RP, mTOR, BAD,
GSK3
MAPK signaling pathway
i.e. MEK, ERK
The range of available assays is rapidly increasing, and an up to date list of
assays is available at the Perkin Elmer website (las.perkinelmer.com).
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The expression level of various targets can vary considerably between cell
lines, as can the level of basal protein phosphorylation during normal cell
growth. Because SureFire assays only use around 4μL of lysate per well, a
typical 96-well or 384-well cell lysate from growing cells be readily assayed for
many targets. Simply lysing a well of growing cells +/- a pathway inhibitor and
analysing for targets of interest can quickly reveal which targets are
detectable along the pathway of interest.
Detection of various phosphoproteins in LNCAP cells
300000
Signal (RFU)
250000
200000
+ wortmannin
150000
No treatment
100000
Caspase 9 p-Ser196
BAD p-Ser112
Akt p-Ser473
p-ERK
4EBP1 p-Thr37/46
S6RP p-Ser235/236
p70S6K p-Thr229
0
GSK3beta p-Ser9
50000
In the experiment above, LNCAP cells were seeded at a density of 25,000
cells per well in a 96-well microplate, and incubated overnight at 37°C/5%
CO2 overnight in RPMI containing 10% FBS, glutamine, sodium pyruvate and
pen/strep. The following morning, the media in the wells was replaced with the
same fresh media, either with or without 2 μM wortmannin, and the cells were
returned to the incubator for a further 3 hours. The media was removed from
the wells and the cells were washed with cold PBS to remove traces of biotin
contained in the media. Finally, the cells were lysed with 50 μL 1X Lysis
Buffer and agitated gently for 10 min. From each culture well, 4 μL lysate was
transferred to 8 different wells of a proxiplate, and analysed for 8 separate
phosphorylation events. Of the 8 targets, 5 showed a usable assay window for
basal phosphorylation in LNCAP cells.
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Common Protocols
General transfer procedure
The most common method for performing these assays is to grow and treat
the cells in a cell culture plate, generate cell lysates, and analyse a portion of
this cell lysate in a separate assay plate transferred from the culture plate.
This ‘transfer’ procedure can be thought of in two stages; (1) the culture of
cells and the stimulation and/or inhibition of particular cellular pathways,
followed by preparation of cellular lysates, and (2) the examination of cellular
lysates for particular phospho-targets of interest.
For adherent cells, there are a number of variations that can be used that are
compatible with SureFire assays. The most common cell culture procedure is
outlined below. Here, cells are plated into microplate wells for an optimal
period of time, followed by the addition of inhibitors and or agonists as
required for an optimal time period. The media can then be removed, and
lysates prepared by the addition of 1X lysis buffer, or the media can remain on
the cells and lysates prepared by the addition of 5X lysis buffer.
Add cells
Adherent cells:
Incubate overnight
Non-adherent cells:
Assay immediately
Add Inhibitors
Adherent cells:
Remove media first or
Add concentrated stock
direct to wells
Non-adherent cells:
Add concentrated stock
direct to wells
Add Stimulant
Adherent cells:
Add concentrated
stock direct to wells
Non-adherent cells:
Add concentrated
stock direct to wells
Lyse cells
Adherent cells:
Remove media first and lyse
with 1X Lysis buffer or
Add 5X Lysis buffer direct to
wells
Non-adherent cells:
Add 5X Lysis buffer direct to
wells
Where possible, we recommend removing the cell culture media prior to cell
lysis. This allows the removal of any agents that are used during the cell
culture experiments that may interfere with the subsequent SureFire assay.
Interfering agents may include compounds that interfere with the antibodytarget interaction or the antibody-protein A interaction, biotin or biotin mimetics
that interfere with the biotin-streptavidin interaction, or free radical scavengers
that interfere with the AlphaScreen bead signal generation.
Once the lysates have been generated, a small aliquot of lysate is transferred
to an assay plate for analysis. The standard protocol recommends transferring
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4 μL of lysate to a low-volume 348-well proxiplate. Because of the low lysate
usage, a typical cell lysate can be analysed for several different targets if
desired.
lysate
Assay Target 1
Assay Target 2
Culture plate
Assay Target 3
Assay plate
Single well procedures
Single well procedures are intended for higher-throughput projects, where
many wells are analysed for a single target. Here the cells are cultured in the
same microplate that they are subsequently assayed in. These may be either
standard white 384-well or 1536-well microplates, or low volume 384-well
proxiplates.
Add cells
Add inhibitors
Add stimulant
Lyse cells
Add assay
reagents
Again, the media may either be removed prior to lysis, or the cells may be
lysed in culture medium by the addition of an appropriate volume of 5X Lysis
buffer. By removing media, the potential for assay interference is reduced.
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Single reagent addition vs dual reagent addition procedures
All SureFire assay kits follow either a procedure with a single simultaneous
reagent addition – the ‘1-step’ procedure, or 2 sequential additions of assay
reagents - the ‘2-step’ procedure. For the 2-step assays, the sensitivity of the
assay is enhanced by separating the reagent additions, a procedure that is
likely to reduce steric constraints of the antibody-antigen complexes, thereby
allowing a more optimal signal to be generated.
2 step assay
Reaction buffer +
Activation buffer +
Acceptor beads
Dilution buffer +
Donor beads
2 hours
1 step assay
Read Plate
Reaction buffer +
Read Plate
Activation buffer +
Both Alphascreen beads
2 hours
2-4 hours
Key features
All assay reagents are added to lysate
simultaneously.
Key features
Assay reagents are added in a stepwise
manner to avoid steric interferences.
Typical assay length 2 x 2 hour
incubations but can be longer
However, for high-throughput users particularly, the ability to add all reagents
simultaneously has the potential to lower liquid handling requirements. For
users that want to minimize liquid handling, most 2-step procedures can be
modified to perform adequately in a one-step format. Here, all assay reagents
- including dilution buffer and donor beads – are mixed together and added to
the lysates simultaneously. A reduction in signal window may be alleviated by
using a longer assay incubation time – for example, overnight incubations can
be used to increase signal window.
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Assay Optimization
There are several parameters that should be optimized to achieve the best
possible assay performance. A useful tool when performing these assays is
the Western blot with an appropriate phospho-antibody. This tool can be used
to determine whether there is a problem with the cells, or with the assay when
an experiment does not perform as expected. As a general guide, if a band
can be seen on a Western blot, then it should be detected with a SureFire
assay also. If not, it may indicate that there may be problems with assay
procedures or kit components.
Selection of assay format
For first time users, we suggest using a 96-well format transfer protocol with
adherent cells, whereas a 384-well single-well assay approach is best when
using non-adherent cells. Once the cell response conditions have been
optimized, it is usually straightforward to transfer the protocol to a more
favourable configuration.
Cell seeding and density
The cell seeding can be very important, depending on which pathway is being
analysed. For example, when looking at ERK phosphorylation, adherent cells
must be seeded in serum-containing media for at least 15 hours (i.e.
overnight) to allow for effective ERK signaling. If cells are seeded in serumfree media, or for shorter periods, the maximal ERK phosphorylation can be
dramatically reduced. Similarly, cell seeding density is important. Typically,
cell number should be titrated to determine an optimal balance between the
assay window, and the amount of cell culture support that is required. The
titration of cell number is also important to determine the responsive range of
the assay. Optimizing cell culture conditions can also have a big influence on
variation induced by factors such as edge-effects and cell clumping leading to
variable cell numbers per well.
Timecourse of activation
Many signaling pathways are not activated in normal growing cells, or are only
activated at low levels. For these pathways, an agonist is often used to
stimulate the cells, inducing a receptor-mediated phosphorylation cascade.
Various proteins that are activated as part of a cascade may take different
times to reach maximal levels of phosphorylation. Further, the maximal
phosphorylation response may be quite transient in some cases, while others
may be induced in a more sustained manner. Therefore, the timecourse of
activation for a particular target of interest must be determined experimentally.
The timecourse will also be influenced on whether the assays are performed
at room temperature or at 37°C.
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Timecourse of NF-κB pathway activation in Hela cells
Phosphorylation (%Max)
120
100
IKKα
80
IKKβ
60
IκB
NF-κB
40
20
0
0
5
10
Time (min)
15
20
In the example above, Hela cells were seeded in 96-well microplates at a
density of 100,000 cells per well, and incubated overnight at 37°C/5% CO2
overnight in MEM containing 10% FBS, sodium pyruvate, non-essential amino
acids and pen/strep. The following morning, the cells were stimulated with
TNFα for varying lengths of time prior to lysis in 50μL of Lysis Buffer. Lysates
were transferred to a proxiplate and assayed for several different kinases
involved in NFκB pathway activation. These experiments demonstrate that
although the kinases analyzed here show quite different timecourse of
phosphorylation, for these experiments a timepoint of 10 min gave a close to
maximal response for all kinases that were analyzed.
Assay buffer system & DMSO tolerance
The SureFire assays are compatible with most cell culture media, however
there are some exceptions. Media that contain biotin (i.e. RPMI) will reduce
assay sensitivity due to the interference of biotin on the antibody-streptavidin
interaction. When it is necessary to use a media such as RPMI for growing
cells, they should be transferred to an alternative media for assaying. When
using non-adherent cells, they can be harvested and resuspended in HBSS or
similar buffers for the assay. For adherent cells this is less of a problem,
because all media can be removed prior to lysis.
DMSO is a common diluent for inhibitor compounds, and is commonly present
in assay buffers at concentrations up to 1%. Because cells can vary in their
tolerance for DMSO, its effect on cell signaling must also be determined. For
example, GPCR-mediated calcium signaling in CHO cells is normally tolerant
of DMSO at 1%, whereas phospho-ERK signaling in the same cells is often
lower than 0.5%.
Maximal response window and basal phosphorylation
For cells with quiescent pathways, the maximal signal window will be
influenced primarily by cell number, lysate concentration, and culturing
techniques such as serum-starvation prior to assay. However, many cells may
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have signaling pathways that exhibit high basal levels of phosphorylation. For
example, many cancer cells may have deregulated PI-3 kinase pathways that
are not lowered by serum starvation or contact inhibition. For these situations,
a pathway inhibitor may often provide a more accurate determination of the
true assay window for a particular cell line. In these cases, an inhibitor will
often drop the background signal to levels that are well below unstimulated
controls.
p70 S6 kinase phosphorylation in PC-3 cells
30000
Inducible above basal with IGF-1
p70S6K p-Thr389 (RFU)
25000
20000
15000
Basal phosphorylation
10000
5000
Complete inhibition
10
-11
10
-10
10
-9
10
-8
10
-7
Log [IGF-1 or Rapamycin] M
In the example above, PC-3 cells were seeded in 96-well microplates at a
density of 50,000 cells per well, and incubated overnight at 37°C/5% CO2
overnight in F12 Kaighn’s media containing 10% FBS, sodium pyruvate, and
pen/strep. The following morning, the cells were either serum starved for 2h,
or treated with various concentrations of rapamycin for 2 hours. Starved cells
were stimulated with IGF-1 for 30 min, and then all wells were lysed in 25μL of
Lysis Buffer. Lysates were transferred to a proxiplate and assayed for p70 S6
kinase phsophorylation at Thr389. Under these conditions, basal
phosphorylation of p70 S6 kinase phsophorylation at Thr389 gives around a
5-fold signal window, which can be approximately doubled by receptormediated pathway activation, using a specific agonist such as IGF-1.
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Troubleshooting
Low Counts
• Ensure the Activation buffer is properly redissolved prior to use.
• Ensure that all assay steps involving AlphaScreen® reagents are
performed in a light-subdued environment. Exposure to bright light can
permanently quench AlphaScreen beads. All bead handling should be
done in either a green light environment, or under low light conditions.
• Ensure that white opaque 384-well low volume microplates (i.e.
proxiplates) are used – the low total assay volume (11 μL) can cause
problems in standard 384 well microplates.
• Ensure incubation temperature for assay is at least 22ºC – temperature
can have a dramatic effect on both antibody binding performance, and
AlphaScreen bead performance.
• Ensure that buffers are prepared correctly. In particular, ensure that 5X
Lysis buffer is diluted to 1X Lysis buffer prior to use, and that the
Activation buffer is fully re-dissolved prior to use.
• Ensure the correct amounts of AlphaScreen beads are used.
• Check that cell density is correct. Cell numbers that are too high or low
can affect the activation of intracellular signaling pathways.
• Ensure cell passage number is not too high, and that cells have not lost
responsiveness.
• A useful guide to the expected kit performance is by analysis using
Western blot. If a target band is observed by Western blot, then a
signal should be detected using the SureFire assay.
High Background
• Check that cell density is correct. Cell numbers that are either too low
or too high can affect basal kinase activation.
• Ensure cell passage number is not too high, and that cells are
behaving as expected.
• Ensure that stimulation buffer did not contain serum if the kinase
pathway that is being monitored is activated by serum.
• Some pathways may have a high level of basal or constitutive activity.
An upstream pathway inhibitor is often useful to determine assay
window for these targets.
• Ensure that AlphaScreen beads are in good condition, and have been
stored and handled correctly.
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Poor Assay Sensitivity
• Ensure the correct amounts of AlphaScreen beads are used.
• Ensure the Activation buffer was properly redissolved prior to use.
• Ensure cell passage number is not too high, and that cells have not lost
responsiveness.
• Ensure incubation temperature for assay is at least 22ºC. Temperature
has a large effect on both the performance of antibody binding, and
also AlphaScreen bead signal.
• Reduce lysis volume to produce more concentrated lysates. Often
endogenous phosphoprotein targets are at low abundance in cells. The
lower the lysis volume, the higher the concentration of the final lysate.
• Use a single-plate method for assaying the target. Transfer methods
typically use only a portion of the total amount of cells that are plated,
whereas single well methods use all of the cells in a particular
experiment.
• A useful guide to expected kit performance is by analysis using
Western blot. If a target band is observed by Western blot, then a
signal should be detected using the SureFire assay.
Poor Cell Stimulation
• Check that the cells are confluent. When confluent, many signaling
pathways – particularly those associated with growth such as MAPK
and PI3K pathways – can become quiescent and synchronized. When
an agonist is introduced to quiescent and synchronized cells, they can
respond uniformly.
• Check that cell density is correct. Too high or low cell numbers can
affect basal kinase activation. Cell density should be such that the
target phosphoprotein is within the responsive range of the assay.
• Ensure cell passage number is not too high, and that cells have lost
responsiveness.
• Check cell harvesting conditions and ensure good cell viability after
harvesting. Typically cells should be maintained in log-phase growth,
and harvested when 70-90% confluent.
• Ensure the kinase pathway of interest is active in the cells, and is
activated by the specific agonist that is used. This may vary depending
on the cell line.
• Ensure that stimulant/agonist is not degraded. Prepare fresh prior to
assay, and use a carrier protein such as BSA if necessary.
Day to Day Variation
• Check cell harvesting conditions, use a standard protocol for cell
culture and harvesting.
• Check for variability in room temperature.
• Check for exposure to bright light sources.
• Check for variation in stimulation times and assay incubation times.
• A useful control for assay variation is to use a standard positive and
negative lysate on all assay plates where possible.
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FAQs
1. What types of cells can be used in the assay?
The assay can be used for many adherent and non-adherent cell types,
including transfected cell lines and primary cells. However, because kinase
expression and phosphorylation conditions can vary from one cell line to
another, parameters such as stimulation time and cell number should be
optimized for each cell line used.
2. What concentration of lysate is required for these assays?
These assays are optimized for the detection of endogenous levels of cellular
kinases. Typically, the SureFire assays work well in the range of 0.2-0.5
mg/mL of lysate. Usually, if a band can be seen on a Western blot for a
particular target, then it should be detectable with the corresponding SureFire
assay. Cells that express low levels of the target of interest, for example if
immunoprecipitation is required to see a positive band on a Western blot, then
it may be below the detectable limit for SureFire assays.
3. Can cell lines with stable or transiently transfected kinase or
receptors be used?
Both transient and stable cell lines have been shown to elicit good
phosphorylation response, however we recommended using stable cell lines
to enhance assay reproducibility. Stable cell lines expressing high levels of a
kinase of interest should give strong signal. However, when using
overexpression systems the concentration of cell lysate should be optimized
to ensure the signal is within the working range of the assay.
4. How should the cells be handled?
Cells should be harvested from flasks for seeding into microplates when
approximately 70-90% confluent. The cells should be detached from the
flasks using mild conditions, accurately counted, and diluted to the
appropriate density in fresh media. If using adherent cells, plate for at least
16h prior to assaying, because adherent cells often require some time to
regain full signaling capacity after harvesting.
5. What parameters require optimization for these assays?
For these assays, several parameters will generally require optimization to
ensure robust and reproducible assay performance. These parameters
include cell seeding density and culturing, whether cells require serum
starvation, and agonist and time course of stimulation. Optimization of
parameters are covered more fully in the optimization section of this
document.
6. Are SureFire assays scalable?
The primary SureFire assay methodology is optimized for a low-volume 384well microplate. It has a total of 11 μL per assay, 4 μL of which is cell lysate,
and 7 μL of assay reagents. However, there are validated protocols that are
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scalable down to 4-5μL total assay volume in 1536-well microplates, allowing
a saving of both lysate and assay reagents. Although the assays can be
scaled up, we do not recommend this due to the excessive use of reagents.
7. Why would I choose a particular assay protocol?
Transfer assay methods are those where the cells are grown in microplates,
typically either 96-well or 384-well, stimulated/inhibited and lysed. A sample of
this lysate is then transferred to an assay plate to analyse for a particular
phosphoprotein. These assays are particularly useful for method development
and optimization, low- to medium-throughput projects, and when assaying for
multiple proteins from a single well. Single plate methods are usually for highthroughput projects, where wells are analysed for a single target, and minimal
use of reagents and liquid handling equipment is essential.
8. Can I assay for multiple targets from a single lysate?
One of the unique features of SureFire protocols is the use of very small
amounts of cell lysate. The standard protocol suggests the use of just 4μL of
lysate per well, whereas a typical 96-well or 384-well cell culture microplate
would use 20-50μL of lysis buffer per well. Therefore, a typical cell lysate can
be assayed for many targets, given temporal and expression level constraints
can vary from cell line to cell line.
9. What is the specificity of these assays?
All SureFire assays react with the human targets. These tend to be well
conserved across species, and most kits can also be used to detect the
corresponding mouse and rat phosphoproteins. Other species should be
tested on a case-by-case basis.
10. Can I incubate my assays overnight?
The assay time is optimized such that the antibody binding is close to
equilibrium after the specified incubation period – usually 2-4 hours when
performing assays at room temperature. However, once equilibrium is
reached, there is no problem in incubating for longer periods, up to overnight
at room temperature, if that is convenient.
11. Can I subtract a background control for data analysis?
In most cases, we would not recommend the subtraction of buffer-only
background during data analysis. For methods such as ELISA, subtraction of
buffers-only controls is possible because cellular debris and interfering
substances are washed away during the many wash steps involved in typical
ELISA protocols. In contrast, SureFire assays are homogeneous, and the
assays are performed and read in crude cellular lysates containing proteins,
lipids, nucleic acids and other cellular debris. Therefore, in this homogeneous
system, the most appropriate background control for subtraction is a cellular
lysate that has no phosphorylated target. Negative lysates can typically be
generated either through the use of kinase inhibitors, and/or non-stimulation
of the assay pathway.
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Contact Details
USA and Europe
Phone:
Please do not hesitate to contact PerkinElmer Customer Care for more
information at toll free 1-800-762-4000 (US & Canada), 0800 111933 (AT),
0800 40858 (B), 800 26588 (L), 808 84236 (DK), 800 117186 (FI), 0805
111333 (F), 0800 1810032 (DE), 800 906642 (I), 0800 234490 (NL), 800
18854 (NW), 800 099164 (SP), 020 0887520 (SE), 0800 000015 (CH), 0800
896046 (GB), 81-45-314-8261 (JP) - Prompt 1 all numbers.
Email:
[email protected] (US and Canada)
[email protected] (Norway, Sweden, Denmark and Finland)
[email protected] (UK and Ireland)
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected] (Belgium, Luxembourg and The Netherlands)
[email protected] (All others)
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC
PROCEDURES.
For more information regarding related SureFire products and protocols
refer to the
PerkinElmer web site: http://las.perkinelmer.com
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